New antibiotics are desperately needed as clinically significant bacterial pathogens have acquired resistance to nearly all existing antibiotics. We describe a high-throughput screening system for identifying new antibacterial agents specifically targeted against an integral component of the bacterial translation apparatus. Work described in this application will identify compounds that selectively inhibit the bacterial initiation factor IF3. We propose to implement a primary screening assay to identify inhibitors of E. coli IF3. The assay uses a reporter fusion system in whole cells and is a parodoxical growth assay. Inhibitors of IF3 are expected to result in increased growth of cells due to higher levels of expression from the reporter gene. Following the primary assay a series of in vitro secondary assays designed to confirm that inhibitors target IF3 function will be performed. Reporter constructs analogous to those used in the primary screening assay will be used in in vitro translation reactions to determine the exact nature of IF3 inhibition. Finally, positive compounds will be evaluated for antibacterial efficacy and for toxicity in mammalian cells. PROPOSED COMMERCIAL APPLICATIONS: This research will develop a new assay to identify novel antibacterial drugs for treating bacterial. infections.